Linear cyclodextrin copolymers

ABSTRACT

Linear cyclodextrin copolymers and linear oxidized cyclodextrin copolymers containing an unoxidized and/or an oxidized cyclodextrin moiety integrated into the polymer backbone are described. Methods of preparing such copolymers are also described. The linear cyclodextrin copolymer and linear oxidized cyclodextrin copolymer of the invention may be used as a delivery vehicle of various therapeutic agents.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional patent application filed under 35 U.S.C. § 121 ofthe copending U.S. application Ser. No. 09/339,818, filed Jun. 25, 1999,which is a continuation-in-part application claiming benefit of priorityunder 35 U.S.C. § 120 to U.S. application Ser. No. 09/203,556, filedDec. 2, 1998, now U.S. Pat. No. 6,509,323 which claims benefit under 35U.S.C. § 119(e) to U.S. Provisional Application Ser. No. 60/091,550,filed Jul. 1, 1998, each of which is herein incorporated by reference inits entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to linear cyclodextrin copolymers and linearoxidized cyclodextrin copolymers. These copolymers, respectively,contain a cyclodextrin moiety, unoxidized or oxidized, as a monomer unitintegrated into the copolymer backbone. The invention also relatesmethods of preparing linear cyclodextrin copolymers and linear oxidizedcyclodextrin copolymers. Such cyclodextrin copolymers may be used as adelivery vehicle of various therapeutic agents.

2. Background of the Invention

Cyclodextrins are cyclic polysaccharides containing naturally occurringD(+)-glucopyranose units in an α-(1,4) linkage. The most commoncyclodextrins are alpha (α)-cyclodextrins, beta (β)-cyclodextrins andgamma (γ)-cyclodextrins which contain, respectively. six, seven or eightglucopyranose units. Structurally, the cyclic nature of a cyclodextrinforms a torus or donut-like shape having an inner apolar or hydrophobiccavity, the secondary hydroxyl groups situated on one side of thecyclodextrin torus and the primary hydroxyl groups situated on theother. Thus, using (β)-cyclodextrin as an example, a cyclodextrin isoften represented schematically as follows:

The side on which the secondary hydroxyl groups are located has a widerdiameter than the side on which the primary hydroxyl groups are located.The hydrophobic nature of the cyclodextrin inner cavity allows for theinclusion of a variety of compounds. (Comprehensive SupramolecularChemistry, Volume 3, J. L. Atwood et al., eds., Pergamon Press (1996);T. Cserhati, Analytical Biochemistry, 225:328-332 (1995); Husain et al.,Applied Spectroscopy, 46:652-658 (1992); FR 2 665 169).

Cyclodextrins have been used as a delivery vehicle of varioustherapeutic compounds by forming inclusion complexes with various drugsthat can fit into the hydrophobic cavity of the cyclodextrin or byforming non-covalent association complexes with other biologicallyactive molecules such as oligonucleotides and derivatives thereof. Forexample, U.S. Pat. No. 4,727,064 describes pharmaceutical preparationsconsisting of a drug with substantially low water solubility and anamorphous, water-soluble cyclodextrin-based mixture. The drug forms aninclusion complex with the Cyclodextrins of the mixture. In U.S. Pat.No. 5,691,316, a cyclodextrin cellular delivery system foroligonucleotides is described. In such a system, an oligonucleotide isnoncovalently complexed with a cyclodextrin or, alternatively, theoligonucleotide may be covalently bound to adamantine which in turn isnon-covalently associated with a cyclodextrin.

Various cyclodextrin containing polymers and methods of theirpreparation are also known in the art. (Comprehensive SupramolecularChemistry, Volume 3, J. L. Atwood et al., eds., Pergamon Press (1996)).A process for producing a polymer containing immobilized cyclodextrin isdescribed in U.S. Pat. No. 5,608,015. According to the process, acyclodextrin derivative is reacted with either an acid halide monomer ofan α,β-unsaturated acid or derivative thereof or with an α,γ-unsaturatedacid or derivative thereof having a terminal isocyanate group or aderivative thereof. The cyclodextrin derivative is obtained by reactingcyclodextrin with such compounds as carbonyl halides and acidanhydrides. The resulting polymer contains cyclodextrin units as sidechains off a linear polymer main chain.

U.S. Pat. No. 5,276,088 describes a method of synthesizing cyclodextrinpolymers by either reacting polyvinyl alcohol or cellulose orderivatives thereof with cyclodextrin derivatives or by copolymerizationof a cyclodextrin derivative with vinyl acetate or methyl methacrylate.Again, the resulting cyclodextrin polymer contains a cyclodextrin moietyas a pendant moiety off the main chain of the polymer.

A biodegradable medicinal polymer assembly with Supramolecular structureis described in WO 96/09073 A1. The assembly comprises a number ofdrug-carrying cyclic compounds prepared by binding a drug to an α, β, orγ-cyclodextrin and then stringing the drug/cyclodextrin compounds alonga linear polymer with the biodegradable moieties bound to both ends ofthe polymer. Such an assembly is reportably capable of releasing a drugin response to a specific biodegradation occurring in a disease. Theseassemblies are commonly referred to as “necklace-type” cyclodextrinpolymers.

However, there still exists a need in the art for linear cyclodextrinpolymers in which the cyclodextrin moiety is part of the main chain andnot a pendant moiety off the main chain and a method for theirpreparation.

SUMMARY OF THE INVENTION

This invention answers this need by providing a linear cyclodextrincopolymer. Such a linear cyclodextrin copolymer has a repeating unit offormula Ia, Ib, or a combination thereof:

The invention also provides methods of preparing a linear cyclodextrincopolymer. One method copolymerizes a cyclodextrin monomer precursordisubstituted with the same or different leaving group and a comonomer Aprecursor capable of displacing the leaving group. Another such methodinvolves iodinating a cyclodextrin monomer precursor to form adiiodinated cyclodextrin monomer precursor and then copolymerizing thediiodinated cyclodextrin monomer precursor with a comonomer A precursorto produce the linear cyclodextrin copolymer. Another method involvesiodinating a cyclodextrin monomer precursor to form a diiodinatedcyclodextrin monomer precursor, aminating the diiodinated cyclodextrinmonomer precursor to form a diaminated cyclodextrin monomer precursorand then copolymerizing the diaminated cyclodextrin monomer precursorwith a comonomer A precursor to produce the linear cyclodextrincopolymer. Yet another method involves the reduction of a linearoxidized cyclodextrin copolymer to the linear cyclodextrin copolymer.

The invention further provides a linear oxidized cyclodextrin copolymer.A linear oxidized cyclodextrin copolymer is a linear cyclodextrincopolymer which contains at least one oxidized cyclodextrin moiety offormula VIa or VIb:

Each cyclodextrin moiety of a linear cyclodextrin copolymer of theinvention may be oxidized so as to form a linear oxidized cyclodextrincopolymer having a repeating unit of formula VIa, VIb, or a combinationthereof.

The invention also provides a method of preparing a linear oxidizedcyclodextrin copolymer. One method involves oxidizing a linearcyclodextrin copolymer such that at least one cyclodextrin monomer isoxidized. Other methods involve copolymerizing an oxidized cyclodextrinmonomer precursor with a comonomer A precursor.

The invention still further provides a linear cyclodextrin copolymer orlinear oxidized cyclodextrin copolymer grafted onto a substrate and amethod of their preparation. The invention also provides a linearcyclodextrin copolymer or linear oxidized cyclodextrin copolymercrosslinked to another polymer and a method of their preparation. Amethod of preparing crosslinked cyclodextrin polymers involves reactinga linear or linear oxidized cyclodextrin copolymer with a polymer in thepresence of a crosslinking agent.

The invention provides a linear cyclodextrin copolymer or linearoxidized cyclodextrin copolymer having at least one ligand bound to thecyclodextrin copolymer. The ligand may be bound to either thecyclodextrin moiety or the comonomer A moiety of the copolymer.

The invention also provides a cyclodextrin composition containing atleast one linear cyclodextrin copolymer of the invention and at leastone linear oxidized cyclodextrin copolymer of the invention. Theinvention also provides therapeutic compositions containing atherapeutic agent and a linear cyclodextrin copolymer and/or a linearoxidized cyclodextrin copolymer of the invention. A method of treatmentby administering a therapeutically effective amount of a therapeuticcomposition of the invention is also described.

DESCRIPTION OF THE DRAWINGS

FIG. 1A shows transfection studies with plasmids encoding Luciferasereporter gene particularly noting the transfection with copolymer 16.

FIG. 1B shows transfection studies with plasmids encoding Luciferasereporter gene particularly noting the toxicity of copolymer 16 toBHK-21.

FIG. 2A shows the effect of copolymer 16/DNA charge ratio and serumconditions on transfection efficiency (● and ▪) and cell survival (▾ and▴) in BHK-21 cells. Result from transfection in 10% serum and serum-freemedia are shown as, respectively, dotted and solid lines. Data arereported at the mean +/− S.D. of three samples. Toxicity data arepresented as best fit lines.

FIG. 2B shows the effect of copolymer 16/DNA charge ratio and serumconditions on transfection efficiency (● and ▪) and cell survival (▾ and▴) in CHO-K1 cells. Results from transfection in 10% serum andserum-free media are shown as, respectively, dotted and solid lines.Data are reported at the mean +/− S.D. of three samples. Toxicity dataare presented as best fit lines.

FIG. 3A shows transfection studies with plasmids encoding Luciferasereporter gene particularly noting the relative light units.

FIG. 3B shows transfection studies with plasmids encoding Luciferasereporter gene particularly noting the fraction cell survival.

DETAILED DESCRIPTION OF THE INVENTION

One embodiment of the invention is a linear cyclodextrin copolymer. Alinear cyclodextrin copolymer is a polymer containing cyclodextrinmoieties as an integral part of its polymer backbone. Previously,cyclodextrin moieties were not a part of the main polymer chain butrather attached off a polymer backbone as pendant moieties.

According to the invention, a linear cyclodextrin copolymer has arepeating unit of formula Ia, Ib, or a combination thereof:

In formula Ia and Ib, C is a substituted or unsubstituted cyclodextrinmonomer and A is a comonomer bound, i.e. covalently bound, tocyclodextrin C. Polymerization of a cyclodextrin monomer C precursorwith a comonomer A precursor results in a linear cyclodextrin copolymerof the invention. Within a single linear cyclodextrin copolymer of theinvention, the cyclodextrin monomer C unit may be the same or differentand, likewise, the comonomer A may be the same or different.

A cyclodextrin monomer precursor may be any cyclodextrin or derivativethereof known in the art. As discussed above, a cyclodextrin is definedas a cyclic polysaccharide most commonly containing six to eightnaturally occurring D(+)-glucopyranose units in an α-(1,4) linkage.Preferably, the cyclodextrin monomer precursor is a cyclodextrin havingsix, seven and eight glucose units, i.e., respectively, an alpha(α)-cyclodextrin, a beta (β)-cyclodextrin and a gamma (γ)-cyclodextrin.A cyclodextrin derivative may be any substituted cyclodextrin known inthe art where the substituent does not interfere with copolymerizationwith comonomer A precursor as described below. According to theinvention, a cyclodextrin derivative may be neutral, cationic oranionic. Examples of suitable substituents include, but are not limitedto, hydroxyalkyl groups, such as, for example, hydroxypropyl,hydroxyethyl; ether groups, such as, for example, dihydroxypropylethers, methyl-hydroxyethyl ethers, ethyl-hydroxyethyl ethers, andethyl-hydroxypropyl ethers; alkyl groups, such as, for example, methyl;saccharides, such as, for example, glucosyl and maltosyl; acid groups,such as, for example, carboxylic acids, phosphorous acids, phosphinousacids, phosphonic acids, phosphoric acids, thiophosphonic acids,thiophosphonic acid and sulfonic acids; imidazole groups; and sulfategroups.

A cyclodextrin monomer precursor may be further chemically modified(e.g. halogenated, aminated) to facilitate or affect copolymerization ofthe cyclodextrin monomer precursor with a comonomer A precursor, asdescribed below. Chemical modification of a cyclodextrin monomerprecursor allows for polymerization at only two positions on eachcyclodextrin moiety, i.e. the creation of a bifunctional cyclodextrinmoiety. The numbering scheme for the C1-C6 positions of eachglucopyranose ring is as follows:

In a preferred embodiment, polymerization occurs at two of any C2, C3and C6 position, including combinations thereof, of the cyclodextrinmoiety. For example, one cyclodextrin monomer precursor may bepolymerized at two C6 positions while another cyclodextrin monomerprecursor may be polymerized at a C2 and a C6 position of thecyclodextrin moiety. Using β-cyclodextrin as an example, the letteringscheme for the relative position of each glucopyranose ring in acyclodextrin is as follows:

In a preferred embodiment of a linear cyclodextrin copolymer of theinvention, the cyclodextrin monomer C has the following general formula(II):

In formula (II), n and m represent integers which, along with the othertwo glucopyranose rings, define the total number of glucopyranose unitsin the cyclodextrin monomer. Formula (II) represents a cyclodextrinmonomer which is capable of being polymerized at two C6 positions on thecyclodextrin unit. Examples of cyclodextrin monomers of formula (II)include, but are not limited to, 6^(A),6^(B)-deoxy-α-cyclodextrin (n=0,m=4), 6^(A),6^(C)-deoxy-α-cyclodextrin (n=1, m=3),6^(A),6^(D)-deoxy-α-cyclodextrin (n=2, m=2),6^(A),6^(B)-deoxy-β-cyclodextrin (n=0, m=5),6^(A),6^(C)-deoxy-β-cyclodextrin (n=1, m=4),6^(A),6^(D)-deoxy-β-cyclodextrin (n=2, m=3),6^(A),6^(B)-deoxy-γ-cyclodextrin (n=0, m=6),6^(A),6^(C)-deoxy-γ-cyclodextrin (n=1, m=5),6^(A),6^(D)-deoxy-γ-cyclodextrin (n=2, m=4), and6^(A),6^(E)-deoxy-γ-cyclodextrin (n=3, m=3). In another preferredembodiment of linear cyclodextrin copolymer of the invention, acyclodextrin monomer C unit has the following general formula (III):

where p=5-7. In formula (III), one of D(+)-glucopyranose units of acyclodextrin monomer has undergone ring opening to allow forpolymerization at a C2 and a C3 position of the cyclodextrin unit.Cyclodextrin monomers of formula (III) are commercially available fromCarbomer of Westborough, Mass. Examples of cyclodextrin monomers offormula (III) include, but are not limited to,2^(A),3^(A)-deoxy-2^(A),3^(A)-dihydro-α-cyclodextrin,2^(A),3^(A)-deoxy-2^(A),3^(A)-dihydro-β-cyclodextrin,2^(A),3^(A)-deoxy-2^(A),3^(A)-dihydro-γ-cyclodextrin, commonly referredto as, respectively, 2,3-deoxy-α-cyclodextrin, 2,3-deoxy-β-cyclodextrin,and 2,3-deoxy-γ-cyclodextrin.

A comonomer A precursor may be any straight chain or branched, symmetricor asymmetric compound which upon reaction with a cyclodextrin monomerprecursor, as described above, links two cyclodextrin monomers together.Preferably, a comonomer A precursor is a compound containing at leasttwo functional groups through which reaction and thus linkage of thecyclodextrin monomers can be achieved. Examples of possible functionalgroups, which may be the same or different, terminal or internal, ofeach comonomer A precursor include, but are not limited to, amino, acid,ester, imidazole, and acyl halide groups and derivatives thereof. In apreferred embodiment, the two functional groups are the same andterminal. Upon copolymerization of a comonomer A precursor with acyclodextrin monomer precursor, two cyclodextrin monomers may be linkedtogether by joining the primary hydroxyl side of one cyclodextrinmonomer with the primary hydroxyl side of another cyclodextrin monomer,by joining the secondary hydroxyl side of one cyclodextrin monomer withthe secondary hydroxyl side of another cyclodextrin monomer, or byjoining the primary hydroxyl side of one cyclodextrin monomer with thesecondary hydroxyl side of another cyclodextrin monomer. Accordingly,combinations of such linkages may exist in the final copolymer. Both thecomonomer A precursor and the comonomer A of the final copolymer may beneutral, cationic (e.g. by containing protonated groups such as, forexample, quaternary ammonium groups) or anionic (e.g. by containingdeprotonated groups, such as, for example, sulfate, phosphate orcarboxylate anionic groups). The charge of comonomer A of the copolymermay be adjusted by adjusting pH conditions. Examples of suitablecomonomer A precursors include, but are not limited to, cystamine,1,6-diaminohexane, diimidazole, dithioimidazole, spermine,dithiospermine, dihistidine, dithiohistidine, succinimide (e.g.dithiobis(succinimidyl propionate) (DSP) and disuccinimidyl suberate(DSS)) and imidates (e.g. dimethyl 3,3′-dithiobispropionimidate (DTBP)).Copolymerization of a comonomer A precursor with a cyclodextrin monomerprecursor leads to the formation of a linear cyclodextrin copolymer ofthe invention containing comonomer A linkages of the following generalformulae:

-   -   —HNC(O)(CH₂)_(x)C(O)NH—, —HNC(O)(CH₂)_(x)SS(CH₂)_(x)C(O)NH—,        —⁺H₂N(CH₂)_(x)SS(CH₂)_(x)NH₂ ⁺—,        —HNC(O)(CH₂CH₂O)_(x)CH₂CH₂C(O)NH—,        —HNNHC(O)(CH₂CH₂O)_(x)CH₂CH₂C(O)NHNH—,        —⁺H₂NCH₂(CH₂CH₂O)_(x)CH₂CH₂CH₂NH₂ ⁺—,        —HNC(O)(CH₂CH₂O)_(x)CH₂CH₂SS(CH₂CH₂O)_(x)CH₂CH₂C(O)NH—, —HNC(NH₂        ⁺)(CH₂CH₂O)_(x)CH₂CH₂C(NH₂ ⁺)NH—, —SCH₂CH₂NHC(NH₂        ⁺)(CH₂)_(x)C(NH₂ ⁺)NHCH₂CH₂S—, —SCH₂CH₂NHC(NH₂        ⁺)(CH₂)_(x)SS(CH₂)_(x)C(NH₂ ⁺)NHCH₂CH₂S—, —SCH₂CH₂NHC(NH₂        ⁺)CH₂CH₂(OCH₂CH₂)_(x)C(NH₂ ⁺)NHCH₂CH₂S—,        In the above formulae, x=1-50, and y+z=x. Preferably, x=1-30.        More preferably, x=1-20. In a preferred embodiment, comonomer A        is biodegradable or acid-labile. Also in a preferred embodiment,        the comonomer A precursor and hence the comonomer A may be        selectively chosen in order to achieve a desired application.        For example, to deliver small molecular therapeutic agents, a        charged polymer may not be necessary and the comonomer A may be        a polyethylene glycol group.

A linear cyclodextrin copolymer of the invention may be modified with atleast one ligand attached to the cyclodextrin copolymer. The ligand maybe attached to the cyclodextrin copolymer through the cyclodextrinmonomer C or comonomer A. Preferably, the ligand is attached to at leastone cyclodextrin moiety of the linear cyclodextrin copolymer.Preferably, the ligand allows a linear cyclodextrin copolymer to targetand bind to a cell. If more than one ligand, which may be the same ordifferent, is attached to a linear cyclodextrin copolymer of theinvention, the additional ligand or ligands may be bound to the same ordifferent cyclodextrin moiety or the same or different comonomer A ofthe copolymer. Examples of suitable ligands include, but are not limitedto, vitamins (e.g. folic acid), proteins (e.g. transferrin, andmonoclonal antibodies) and polysaccharides. The ligand will varydepending upon the type of delivery desired. For example,receptor-mediated delivery may by achieved by, but not limited to, theuse of a folic acid ligand while antisense oligo delivery may beachieved by, but not limited to, use of a transferrin ligand. The ligandmay be attached to a copolymer of the invention by means known in theart.

Another embodiment of the invention is a method of preparing a linearcyclodextrin copolymer. According to the invention, a linearcyclodextrin copolymer of the invention may be prepared bycopolymerizing a cyclodextrin monomer precursor disubstituted with anappropriate leaving group with a comonomer A precursor capable ofdisplacing the leaving groups. The leaving group, which may be the sameor different, may be any leaving group known in the art which may bedisplaced upon copolymerization with a comonomer A precursor. In apreferred embodiment, a linear cyclodextrin copolymer may be prepared byiodinating a cyclodextrin monomer precursor to form a diiodinatedcyclodextrin monomer precursor and copolymerizing the diiodinatedcyclodextrin monomer precursor with a comonomer A precursor to form alinear cyclodextrin copolymer having a repeating unit of formula Ia, Ib,or a combination thereof, each as described above. In a preferredembodiment, a method of preparing a linear cyclodextrin of the inventioniodinates a cyclodextrin monomer precursor as described above to form adiiodinated cyclodextrin monomer precursor of formula IVa, IVb, IVc or amixture thereof:

The diiodinated cyclodextrin may be prepared by any means known in theart. (Tabushi et al. J. Am. Chem. 106, 5267-5270 (1984); Tabushi et al.J. Am. Chem. 106, 4580-4584 (1984)). For example, β-cyclodextrin may bereacted with biphenyl-4,4′-disulfonyl chloride in the presence ofanhydrous pyridine to form a biphenyl-4,4′-disulfonyl chloride cappedβ-cyclodextrin which may then be reacted with potassium iodide toproduce diiodo-β-cyclodextrin. The cyclodextrin monomer precursor isiodinated at only two positions. By copolymerizing the diiodinatedcyclodextrin monomer precursor with a comonomer A precursor, asdescribed above, a linear cyclodextrin polymer having a repeating unitof formula Ia, Ib, or a combination thereof, also as described above,may be prepared. If appropriate, the iodine or iodo groups may bereplaced with other known leaving groups.

Also according to the invention, the iodo groups or other appropriateleaving group may be displaced with a group that permits reaction with acomonomer A precursor, as described above. For example, a diiodinatedcyclodextrin monomer precursor of formula IVa, IVb, IVc or a mixturethereof may be aminated to form a diaminated cyclodextrin monomerprecursor of formula Va, Vb, Vc or a mixture thereof:

The diaminated cyclodextrin monomer precursor may be prepared by anymeans known in the art. (Tabushi et al. Tetrahedron Lett. 18:1527-1530(1977); Mungall et al., J. Org. Chem. 1659-1662 (1975)). For example, adiiodo-β-cyclodextrin may be reacted with sodium azide and then reducedto form a diamino-β-cyclodextrin. The cyclodextrin monomer precursor isaminated at only two positions. The diaminated cyclodextrin monomerprecursor may then be copolymerized with a comonomer A precursor, asdescribed above, to produce a linear cyclodextrin copolymer having arepeating unit of formula Ia, Ib, or a combination thereof, also asdescribed above. However, the amino functionality of a diaminatedcyclodextrin monomer precursor need not be directly attached to thecyclodextrin moiety. Alternatively, the amino functionality may beintroduced by displacement of the iodo or other appropriate leavinggroups of a cyclodextrin monomer precursor with amino group containingmoieties such as, for example, —SCH₂CH₂NH₂, to form a diaminatedcyclodextrin monomer precursor of formula Vd, Ve, Vf or a mixturethereof:

A linear cyclodextrin copolymer of the invention may also be prepared byreducing a linear oxidized cyclodextrin copolymer of the invention asdescribed below. This method may be performed as long as the comonomer Adoes not contain a reducible moiety or group such as, for example, adisulfide linkage.

According to the invention, a linear cyclodextrin copolymer of theinvention may be oxidized so as to introduce at least one oxidizedcyclodextrin monomer into the copolymer such that the oxidizedcyclodextrin monomer is an integral part of the polymer backbone. Alinear cyclodextrin copolymer which contains at least one oxidizedcyclodextrin monomer is defined as a linear oxidized cyclodextrincopolymer. The cyclodextrin monomer may be oxidized on either thesecondary or primary hydroxyl side of the cyclodextrin moiety. If morethan one oxidized cyclodextrin monomer is present in a linear oxidizedcyclodextrin copolymer of the invention, the same or differentcyclodextrin monomers oxidized on either the primary hydroxyl side, thesecondary hydroxyl side, or both may be present. For illustrationpurposes, a linear oxidized cyclodextrin copolymer with oxidizedsecondary hydroxyl groups has, for example, at least one unit of formulaVIa or VIb:

In formulae VIa and VIb, C is a substituted or unsubstituted oxidizedcyclodextrin monomer and A is a comonomer bound, i.e. covalently bound,to the oxidized cyclodextrin C. Also in formulae VIa and VIb, oxidationof the secondary hydroxyl groups leads to ring opening of thecyclodextrin moiety and the formation of aldehyde groups.

A linear oxidized cyclodextrin copolymer may be prepared by oxidation ofa linear cyclodextrin copolymer as discussed above. Oxidation of alinear cyclodextrin copolymer of the invention may be accomplished byoxidation techniques known in the art. (Hisamatsu et al., Starch44:188-191 (1992)). Preferably, an oxidant such as, for example, sodiumperiodate is used. It would be understood by one of ordinary skill inthe art that under standard oxidation conditions that the degree ofoxidation may vary or be varied per copolymer. Thus in one embodiment ofthe invention, a linear oxidized copolymer of the invention may containone oxidized cyclodextrin monomer. In another embodiment, substantiallyall to all cyclodextrin monomers of the copolymer would be oxidized.

Another method of preparing a linear oxidized cyclodextrin copolymer ofthe invention involves the oxidation of a diiodinated or diaminatedcyclodextrin monomer precursor, as described above, to form an oxidizeddiiodinated or diaminated cyclodextrin monomer precursor andcopolymerization of the oxidized diiodinated or diaminated cyclodextrinmonomer precursor with a comonomer A precursor. In a preferredembodiment, an oxidized diiodinated cyclodextrin monomer precursor offormula VIIa, VIIb, VIIc, or a mixture thereof:

may be prepared by oxidation of a diiodinated cyclodextrin monomerprecursor of formulae IVa, IVb, IVc, or a mixture thereof, as describedabove. In another preferred embodiment, an oxidized diaminatedcyclodextrin monomer precursor of formula VIIIa, VIIIb, VIIIc or amixture thereof:

may be prepared by amination of an oxidized diiodinated cyclodextrinmonomer precursor of formulae VIIa, VIIb, VIIc, or a mixture thereof, asdescribed above. In still another preferred embodiment, an oxidizeddiaminated cyclodextrin monomer precursor of formula IXa, IXb, IXc or amixture thereof:

may be prepared by displacement of the iodo or other appropriate leavinggroups of an oxidized cyclodextrin monomer precursor disubstituted withan iodo or other appropriate leaving group with the amino groupcontaining moiety —SCH₂CH₂NH₂.

Alternatively, an oxidized diiodinated or diaminated cyclodextrinmonomer precursor, as described above, may be prepared by oxidizing acyclodextrin monomer precursor to form an oxidized cyclodextrin monomerprecursor and then diiodinating and/or diaminating the oxidizedcyclodextrin monomer, as described above. As discussed above, thecyclodextrin moiety may be modified with other leaving groups other thaniodo groups and other amino group containing functionalities. Theoxidized diiodinated or diaminated cyclodextrin monomer precursor maythen be copolymerized with a comonomer A precursor, as described above,to form a linear oxidized cyclodextrin copolymer of the invention.

A linear oxidized cyclodextrin copolymer may also be further modified byattachment of at least one ligand to the copolymer. The ligand is asdescribed above.

In a preferred embodiment of the invention, a linear cyclodextrincopolymer or a linear oxidized cyclodextrin copolymer terminates with atleast one comonomer A precursor or hydrolyzed product of the comonomer Aprecursor, each as described above. As a result of termination of thecyclodextrin copolymer with at least one comonomer A precursor, at leastone free functional group, as described above, exists per linearcyclodextrin copolymer or per linear oxidized cyclodextrin copolymer.For example, the functional group may be an acid group or a functionalgroup that may be hydrolyzed to an acid group. According to theinvention, the functional group may be further chemically modified asdesired to enhance the properties of the cyclodextrin copolymer, suchas, for example, colloidal stability, and transfection efficiency. Forexample, the functional group may be modified by reaction with PEG toform a PEG terminated cyclodextrin copolymer to enhance colloidalstability or with histidine to form an imidazolyl terminatedcyclodextrin copolymer to enhance intracellular and transfectionefficiency.

Further chemistry may be performed on the cyclodextrin copolymer throughthe modified functional group. For example, the modified functionalgroup may be used to extend a polymer chain by linking a linearcyclodextrin copolymer or linear oxidized cyclodextrin copolymer, asdescribed herein, to the same or different cyclodextrin copolymer or toa non-cyclodextrin polymer. In a preferred embodiment of the invention,the polymer to be added on is the same or different linear cyclodextrincopolymer or linear oxidized cyclodextrin copolymer which may alsoterminated with at least one comonomer A precursor for furthermodification, each as described herein.

Alternatively, at least two of the same or different linear cyclodextrincopolymers or linear oxidized cyclodextrin copolymers containing aterminal functional group or a terminal modified functional group, asdescribed above, may be reacted and linked together through thefunctional or modified functional group. Preferably, upon reaction ofthe functional or modified functional groups, a degradable moiety suchas, for example, a disulfide linkage is formed. For example,modification of the terminal functional group with cysteine may be usedto produce a linear cyclodextrin copolymer or linear oxidizedcyclodextrin copolymer having at least one free thiol group. Reactionwith the same or different cyclodextrin copolymer also containing atleast one free thiol group will form a disulfide linkage between the twocopolymers. In a preferred embodiment of the invention, the functionalor modified functional groups may be selected to offer linkagesexhibiting different rates of degradation (e.g. via enzymaticdegradation) and thereby provide, if desired, a time release system fora therapeutic agent. The resulting polymer may be crosslinked, asdescribed herein. A therapeutic agent, as described herein, may be addedprior to or post crosslinking of the polymer. A ligand, as describedherein, may also be bound through the modified functional group.

According to the invention, a linear cyclodextrin copolymer or linearoxidized cyclodextrin copolymer may be attached to or grafted onto asubstrate. The substrate may be any substrate as recognized by those ofordinary skill in the art. In another preferred embodiment of theinvention, a linear cyclodextrin copolymer or linear oxidizedcyclodextrin copolymer may be crosslinked to a polymer to form,respectively, a crosslinked cyclodextrin copolymer or a crosslinkedoxidized cyclodextrin copolymer. The polymer may be any polymer capableof crosslinking with a linear or linear oxidized cyclodextrin copolymerof the invention (e.g. polyethylene glycol (PEG) polymer, polyethylenepolymer). The polymer may also be the same or different linearcyclodextrin copolymer or linear oxidized cyclodextrin copolymer. Thus,for example, a linear cyclodextrin copolymer may be crosslinked to anypolymer including, but not limited to, itself, another linearcyclodextrin copolymer, and a linear oxidized cyclodextrin copolymer. Acrosslinked linear cyclodextrin copolymer of the invention may beprepared by reacting a linear cyclodextrin copolymer with a polymer inthe presence of a crosslinking agent. A crosslinked linear oxidizedcyclodextrin copolymer of the invention may be prepared by reacting alinear oxidized cyclodextrin copolymer with a polymer in the presence ofan appropriate crosslinking agent. The crosslinking agent may be anycrosslinking agent known in the art. Examples of crosslinking agentsinclude dihydrazides and disulfides. In a preferred embodiment, thecrosslinking agent is a labile group such that a crosslinked copolymermay be uncrosslinked if desired.

A linear cyclodextrin copolymer and a linear oxidized cyclodextrincopolymer of the invention may be characterized by any means known inthe art. Such characterization methods or techniques include, but arenot limited to, gel permeation chromatography (GPC), matrix assistedlaser desorption ionization-time of flight mass spectrometry (MALDI-TOFMass spec), ¹H and ¹³C NMR, light scattering and titration.

The invention also provides a cyclodextrin composition containing atleast one linear cyclodextrin copolymer and at least one linear oxidizedcyclodextrin copolymer of the invention as described above. Accordingly,either or both of the linear cyclodextrin copolymer and linear oxidizedcyclodextrin copolymer may be crosslinked to another polymer and/orbound to a ligand as described above. Therapeutic compositions accordingto the invention contain a therapeutic agent and a linear cyclodextrincopolymer or a linear oxidized cyclodextrin copolymer, includingcrosslinked copolymers, of the invention. A linear cyclodextrincopolymer, a linear oxidized cyclodextrin copolymer and theircrosslinked derivatives are as described above. The therapeutic agentmay be any synthetic or naturally occurring biologically activetherapeutic agent including those known in the art. Examples of suitabletherapeutic agents include, but are not limited to, antibiotics,steroids, polynucleotides (e.g. genomic DNA, cDNA, mRNA and antisenseoligonucleotides), plasmids, peptides, peptide fragments, smallmolecules (e.g. doxorubicin) and other biologically activemacromolecules such as, for example, proteins and enzymes.

A therapeutic composition of the invention may be prepared by meansknown in the art. In a preferred embodiment, a copolymer of theinvention is mixed with a therapeutic agent, as described above, andallowed to self-assemble. According to the invention, the therapeuticagent and a linear cyclodextrin copolymer or a linear oxidizedcyclodextrin copolymer of the invention associate with one another suchthat the copolymer acts as a delivery vehicle for the therapeutic agent.The therapeutic agent and cyclodextrin copolymer may associate by meansrecognized by those of skill in the art such as, for example,electrostatic interaction and hydrophobic interaction. The degree ofassociation may be determined by techniques known in the art including,for example, fluorescence studies, DNA mobility studies, lightscattering, electron microscopy, and will vary depending upon thetherapeutic agent. As a mode of delivery, for example, a therapeuticcomposition of the invention containing a copolymer of the invention andDNA may be used to aid in transfection, i.e. the uptake of DNA into ananimal (e.g. human) cell. (Boussif, O. Proceedings of the NationalAcademy of Sciences, 92:7297-7301 (1995); Zanta et al. BioconjugateChemistry, 8:839-844 (1997)).

A therapeutic composition of the invention may be, for example, a solid,liquid, suspension, or emulsion. Preferably a therapeutic composition ofthe invention is in a form that can be injected intravenously. Othermodes of administration of a therapeutic composition of the inventioninclude, depending on the state of the therapeutic composition, methodsknown in the art such as, but not limited to, oral administration,topical application, parenteral, intravenous, intranasal, intraocular,intracranial or intraperitoneal injection.

Depending upon the type of therapeutic agent used, a therapeuticcomposition of the invention may be used in a variety of therapeuticmethods (e.g. DNA vaccines, antibiotics, antiviral agents) for thetreatment of inherited or acquired disorders such as, for example,cystic fibrosis, Gaucher's disease, muscular dystrophy, AIDS, cancers(e.g. multiple myeloma, leukemia, melanoma, and ovarian carcinoma),cardiovascular conditions (e.g., progressive heart failure, restenosis,and hemophilia), and neurological conditions (e.g. brain trauma).According to the invention, a method of treatment administers atherapeutically effective amount of a therapeutic composition of theinvention. A therapeutically effective amount, as recognized by those ofskill in the art, will be determined on a case by case basis. Factors tobe considered include, but are not limited to, the disorder to betreated and the physical characteristics of the one suffering from thedisorder.

Another embodiment of the invention is a composition containing at leastone biologically active compound having agricultural utility and alinear cyclodextrin copolymer or a linear oxidized cyclodextrincopolymer of the invention. The agriculturally biologically activecompounds include those known in the art. For example, suitableagriculturally biologically active compounds include, but are notlimited to, fungicides, herbicides, insecticides, and mildewcides.

The following examples are given to illustrate the invention. It shouldbe understood, however, that the invention is not to be limited to thespecific conditions or details described in these examples.

EXAMPLES

Materials. β-cyclodextrin (Cerestar USA, Inc. of Hammond, Ind.) wasdried in vacuo (<0.1 mTorr) at 120° C. for 12 h before use.Biphenyl-4,4′-disulfonyl chloride (Aldrich Chemical Company, Inc. ofMilwaukee, Wis.) was recrystallized from chloroform/hexanes. Potassiumiodide was powdered with a mortar and pestle and dried in an oven at200° C. All other reagents were obtained from commercial suppliers andwere used as received without further purification. Polymer samples wereanalyzed on a Hitachi HPLC system equipped with an Anspec RI detectorand a Progel-TSK G3000_(PWXL) column using water as eluant at a 1.0 mLmin⁻¹ flow rate.

Example 1

Biphenyl-4,4′-disulfonyl-A,D-Capped β-Cyclodextrin, 1 (Tabushi et al. J.Am. Chem. Soc. 106, 5267-5270 (1984))

A 500 mL round bottom flask equipped with a magnetic stirbar, a Schlenkadapter and a septum was charged with 7.92 g (6.98 mmol) of dryβ-cyclodextrin and 250 mL of anhydrous pyridine (Aldrich ChemicalCompany, Inc.). The resulting solution was stirred at 50° C. undernitrogen while 2.204 g (6.28 mmol) of biphenyl-4,4′-disulfonyl chloridewas added in four equal portions at 15 min intervals. After stirring at50 ° C. for an additional 3 h, the solvent was removed in vacuo and theresidue was subjected to reversed-phase column chromatography using agradient elution of 0-40% acetonitrile in water. Fractions were analyzedby high performance liquid chromatography (HPLC) and the appropriatefractions were combined. After removing the bulk of the acetonitrile ona rotary evaporator, the resulting aqueous suspension was lyophilized todryness. This afforded 3.39 g (38%) of 1 as a colorless solid.

Example 2

6^(A),6^(D)-Diiodo-6^(A),6^(D)-Deoxy-β-cyclodextrin, 2 (Tabushi et al.J. Am. Chem. 106, 4580-4584 (1984))

A 40 mL centrifuge tube equipped with a magnetic stirbar, a Schlenkadapter and a septum was charged with 1.02 g (7.2 mmol) of 1, 3.54 g(21.3 mmol) of dry, powdered potassium iodide (Aldrich) and 15 mL ofanhydrous N,N-dimethylformamide (DMF) (Aldrich). The resultingsuspension was stirred at 80° C. under nitrogen for 2 h. After coolingto room temperature, the solids were separated by centrifugation and thesupernatant was decanted. The solid precipitate was washed with a secondportion of anhydrous DMF and the supernatants were combined andconcentrated in vacuo. The residue was then dissolved in 14 mL of waterand cooled in an ice bath before 0.75 mL (7.3 mmol) oftetrachloroethylene (Aldrich) was added with rapid stirring. Theprecipitated inclusion complex was filtered on a medium glass frit andwashed with a small portion of acetone before it was dried under vacuumover P₂O₅ for 14 h. This afforded 0.90 g (92%) of 2 as a white solid.

Example 3

6^(A),6^(D)-Diazido-6^(A),6^(D)-Deoxy-β-cyclodextrin, 3 (Tabushi et al.Tetrahedron Lett. 18, 1527-1530 (1977))

A 100 mL round bottom flask equipped with a magnetic stirbar, a Schlenkadapter and a septum was charged with 1.704 g (1.25 mmol) ofβ-cyclodextrin diiodide, 0.49 g (7.53 mmol) of sodium azide (EM Scienceof Gibbstown, N.J.) and 10 mL of anhydrous N,N-dimethylformamide (DMF).The resulting suspension was stirred at 60° C. under nitrogen for 14 h.The solvent was then removed in vacuo. The resulting residue wasdissolved in enough water to make a 0.2 M solution in salt and thenpassed through 11.3 g of Biorad AG501-X8(D) resin to remove residualsalts. The eluant was then lyophilized to dryness yielding 1.232 g (83%)of 3 as a white amorphous solid which was carried on to the next stepwithout further purification.

Example 4

6^(A),6^(D)-Diamino-6^(A),6^(D)-Deoxy-β-cyclodextrin, 4 (Mungall et al.,J. Org. Chem. 1659-1662 (1975))

A 250 mL round bottom flask equipped with a magnetic stirbar and aseptum was charged with 1.232 g (1.04 mmol) of β-cyclodextrin bisazideand 50 mL of anhydrous pyridine (Aldrich). To this stirring suspensionwas added 0.898 g (3.42 mmol) of triphenylphosphine. The resultingsuspension was stirred for 1 h at ambient temperature before 10 mL ofconcentrated aqueous ammonia was added. The addition of ammonia wasaccompanied by a rapid gas evolution and the solution becamehomogeneous. After 14 h, the solvent was removed in vacuo and theresidue was triterated with 50 mL of water. The solids were filtered offand the filtrate was made acidic (pH<4) with 10% HCl before it wasapplied to an ion exchange column containing Toyopearl SP-650M (NH₄⁺form) resin. The product 4 was eluted with a gradient of 0-0.5 Mammonium bicarbonate. Appropriate fractions were combined andlyophilized to yield 0.832 g (71%) of the product 4 as the bis(hydrogencarbonate) salt.

Example 5

β-cyclodextrin-DSP copolymer, 5

A 20 mL scintillation vial was charged with a solution of 92.6 mg(7.65×10⁻⁵ mol) of the bis(hydrogen carbonate) salt of 4 in 1 mL ofwater. The pH of the solution was adjusted to 10 with 1 M NaOH before asolution of 30.9 mg (7.65×10⁻⁵ mol) of dithiobis(succinimidylpropionate) (DSP, Pierce Chemical Co. of Rockford, Ill.) in 1 mL ofchloroform was added. The resulting biphasic mixture was agitated with aVortex mixer for 0.5 h. The aqueous layer was then decanted andextracted with 3×1 mL of fresh chloroform. The aqueous polymer solutionwas then subjected to gel permeation chromatography (GPC) on ToyopearlHW-40F resin using water as eluant. Fractions were analyzed by GPC andappropriate fractions were lyophilized to yield 85 mg (85%) as acolorless amorphous powder.

Example 6

β-cyclodextrin-DSS copolymer, 6

A β-cyclodextrin-DSS copolymer, 6, was synthesized in a manner analogousto the DSP polymer, 5, except that disuccinimidyl suberate (DSS, PierceChemical Co. of Rockford, Ill.) was substituted for the DSP reagent.Compound 6 was obtained in 67% yield.

Example 7

β-cyclodextrin-DTBP copolymer, 7

A 20 mL scintillation vial was charged with a solution of 91.2 mg(7.26×10⁻⁵ mol) of the bis(hydrogen carbonate) salt of 4 in 1 mL ofwater. The pH of the solution was adjusted to 10 with 1 M NaOH before22.4 mg (7.26×10⁻⁵ mol) of dimethyl 3,3′-dithiobis(propionimidate).2 HCl(DTBP, Pierce Chemical Co. of Rockford, Ill.) was added. The resultinghomogeneous solution was agitated with a Vortex mixer for 0.5 h. Theaqueous polymer solution was then subjected to gel permeationchromatography (GPC) on Toyopearl HW-40F resin. Fractions were analyzedby GPC and appropriate fractions were lyophilized to yield 67 mg (67%)of a colorless amorphous powder.

Example 8

β-cyclodextrin-cystamine copolymer, 8

To a solution of 166.2 mg (7.38×10⁻⁵ mol) of cystamine dihydrochloride(Aldrich) in 15 mL of 0.1 N NaOH was added 100 mg (7.38×10⁻⁵ mol) of 2and 5 mL of acetonitrile. The resulting homogeneous solution was heatedat 80° C. for 2 h before it was subjected to gel permeationchromatography (GPC) on Toyopearl HW-40F resin. Fractions were analyzedby GPC and appropriate fractions were lyophilized to yield 17.2 mg (19%)of a colorless amorphous powder.

Example 9

Polyethylene glycol 600 dihydrazide, 9

A 100 mL round bottom flask equipped with a magnetic stirbar and areflux condenser was charged with 1.82 g (3.0 mmol) of polyethyleneglycol 600 (Fluka Chemical Corp of Milwaukee, Wis.), 40 mL of absoluteethanol (Quantum Chemicals Pty Ltd of Tuscola, Ill.) and a few drops ofsulfuric acid. The resulting solution was heated to reflux for 14 h.Solid sodium carbonate was added to quench the reaction and the solutionof the PEG diester was transferred under nitrogen to an addition funnel.This solution was then added dropwise to a solution of 0.6 mL (9.0 mmol)of hydrazine hydrate (Aldrich) in 10 mL of absolute ethanol. A smallamount of a cloudy precipitate formed. The resulting solution was heatedto reflux for 1 h before it was filtered and concentrated. GPC analysisrevealed a higher molecular weight impurity contaminating the product.Gel permeation chromatography on Toyopearl HW-40 resin enabled a partialpurification of this material to approximately 85% purity.

Example 10

Oxidation of β-cyclodextrin-DSS copolymer, 10 (Hisamatsu et al., Starch44, 188-191 (1992))

The β-cyclodextrin-DSS copolymer 6 (92.8 mg, 7.3×10⁻⁵ mol) was dissolvedin 1.0 mL of water and cooled in an ice bath before 14.8 mg (7.3×10⁻⁵mol) of sodium periodate was added. The solution immediately turnedbright yellow and was allowed to stir in the dark at 0° C. for 14 h. Thesolution was then subjected to gel permeation chromatography (GPC) onToyopearl HW-40 resin using water as eluant. Fractions were analyzed byGPC. Appropriate fractions were combined and lyophilized to dryness toyield 84.2 mg (91%) of a light brown amorphous solid.

Example 11

Polyethylene glycol (PEG) 600 diacid chloride, 11

A 50 mL round bottom flask equipped with a magnetic stirbar and a refluxcondenser was charged with 5.07 g (ca. 8.4 mmol) of polyethylene glycol600 diacid (Fluka Chemical Corp of Milwaukee, Wis.) and 10 mL ofanhydrous chloroform (Aldrich). To this stirring solution was added 3.9mL (53.4 mmol) of thionyl chloride (Aldrich) and the resulting solutionwas heated to reflux for 1 h, during which time gas evolution wasevident. The resulting solution was allowed to cool to room temperaturebefore the solvent and excess thionyl chloride were removed in vacuo.The resulting oil was stored in a dry box and used without purification.

Example 12

β-cyclodextrin-PEG 600 copolymer, 12

A 20 mL scintillation vial was charged with a solution of 112.5 mg(8.95×10⁻⁵ mol) of the bis(hydrogen carbonate) salt of6^(A),6^(D)-diamino-6^(A),6^(D)-deoxy-β-cyclodextrin, 50 μL (3.6×10⁻⁴mol) of triethylamine (Aldrich), and 5 mL of anhydrousN,N-dimethylacetamide (DMAc, Aldrich). The resulting suspension was thentreated with 58 mg (9.1×10⁻⁵ mol) of polyethylene glycol 600 diacidchloride, 11. The resulting solution was agitated with a Vortex mixerfor 5 minutes and then allowed to stand at 25° C. for 1 h during whichtime it became homogeneous. The solvent was removed in vacuo and theresidue was subjected to gel permeation chromatography on ToyopearlHW-40F resin using water as eluant. Fractions were analyzed by GPC andappropriate fractions were lyophilized to dryness to yield 115 mg (75%)of a colorless amorphous powder.

Example 13

β-cyclodextrin-DSP copolymer, 13

A 8 mL vial was charged with a solution of 102.3 mg (8.80×10−5 mol) of2^(A),3^(A)-diamino-2^(A),3^(A)-deoxy-β-cyclodextrin in 1 mL of water.The pH of the solution was adjusted to 10 with 1 M NaOH before asolution of 36.4 mg (8.80×10⁻⁵ mol) of dithiobis(succinimidylpropionate) (DSP, Pierce Chemical Co. of Rockford, Ill.) in 1 mL ofchloroform was added. The resulting biphasic mixture was agitated with aVortex mixer for 0.5 h. The aqueous layer was then decanted andextracted with 3×1 mL of fresh chloroform. The aqueous polymer solutionwas then subjected to gel permeation chromatography.

Example 14

6^(A),6^(D)-Bis-(2-aminoethylthio)-6^(A)6^(D)-deoxy-B-cyclodextrin, 14(Tabushi, I: Shimokawa, K; Fugita, K. Tetrahedron Lett. 1977, 1527-1530)

A 25 mL Schlenk flask equipped with a magnetic stirbar and a septum wascharged with 0.91 mL (7.37 mmol) of a 0.81 M solution of sodium2-aminoethylthiolate in ethanol. (Fieser, L. F.; Fiester, M. Reagentsfor Organic Synthesis; Wiley: New York, 1967; Vol. 3, pp. 265-266). Thesolution was evaporated to dryness and the solid was redissolved in 5 mLof anhydrous DMF (Aldrich).6^(A),6^(D)-Diiodo-6^(A),6^(D)-deoxy-β-cyclodextrin (100 mg, 7.38×10⁻⁵mol was added and the resulting suspension was stirred at 60° C. undernitrogen for 2 h. After cooling to room temperature, the solution wasconcentrated in vacuo and the residue was redissolved in water. Afteracidifying with 0.1 N HCl, the solution was applied to a ToyopearlSP-650M ion-exchange column (NH₄ ⁺ form) and the product was eluted witha 0 to 0.4 M ammonium bicarbonate gradient. Appropriate fractions werecombined and lyophilized to dryness. This afforded 80 mg (79%) of 14 asa white powder.

Example 15

β-cyclodextrin(cystamine)-DTBP copolymer, 15

A 4 mL vial was charged with a solution of 19.6 mg (1.42×10⁻⁵ mol) ofthe bis(hydrogen carbonate) salt of 14 in 0.5 mL of 0.1 M NaHCO₃. Thesolution was cooled in an ice bath before 4.4 mg (1.4×10⁻⁵ mol) ofdimethyl 3,3′-dithiobispropionimidate-2 HCl (DTBP, Pierce) was added.The resulting solution was then agitated with a Vortex mixer and allowedto stand at 0° C. for 1 h. The reaction was quenched with 1M Tris-HClbefore it was acidified to pH 4 with 0.1 N HCl. The aqueous polymersolution was then subjected to gel permeation chromatography onToyopearl HW-40F resin. Fractions were analyzed by GPC and appropriatefractions were lyophilized to dryness. This afforded 21.3 mg (100%) of15 as a white powder.

Example 16

β-cyclodextrin(cystamine)-DMS copolymer, 16

A 10 mL Schlenk flask equipped with a magnetic stirbar and a septum wascharged with 200 mg (1.60×10⁻⁴ mol) of 14, 44 μL (3.2×10⁻⁴ mol) oftriethylamine (Aldrich Chemical Co., Milwaukee, Wis.), 43.6 mg(1.60×10⁻⁴ mol) of dimethylsuberimidate.2HCl (DMS, Pierce), and 3 mL ofanhydrous DMF (Aldrich Chemical Co., Milwaukee, Wis.). The resultingslurry was heated to 80° C. for 18 hours under a steady stream ofnitrogen during which time most of the solvent had evaporated. Theresidue which remained was redissolved in 10 mL of water and theresulting solution was then acidified with 10% HCl to pH 4. Thissolution was then passed through an Amicon Centricon Plus-20 5,000 NMWLcentrifugal filter. After washing with 2×10 mL portions of water, thepolymer solution was lyophilized to dryness yielding 41.4 mg (18%) of anoff-white amorphous solid.

Example 17

Folate Ligand Attachment to Cyclodextrin Polymer

1. Resin Coupling

50 mg of FMOC-PEG₃₄₀₀-NHS (Shearwater Polymers, Inc. of Huntsville,Ala.) is dissolved in 1 mL of anhydrous N,N-dimethylformamide (DMF) andis added to 10 equivalents of hydrazide 2-chlorotrityl resin(Novabiochem USA of La Jolla, Calif.) swelled in DMF. The mixture isstirred at 60° C. until all the polymer is coupled to the resin, asdetermined by a GPC system equipped with a UV detector. Theresin-polymer is then transferred to a sintered glass column for allfurther reactions.

2. Resin Capping

The unreacted hydrazide groups on the resins are capped with aceticanhydride and the acetic acid products are neutralized bydiisopropylethylamine.

3. Removal of Protecting Group

The FMOC protecting group is removed by two washes with 20% piperidinein DMF (1 mL total volume). The resin is then washed 10 times with 1 mLDMF and 5 times with 1 mL H₂O.

4. Folic Acid Coupling

10 equivalents of folic acid and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) is added to theresin along with 1.5 mL H₂O. 1N NaOH is added to the reaction mixtureuntil the folic acid is dissolved (around pH 10). The glass column isthen placed on a rotator and mixed overnight. The resin is then washed10 times with 1 mL NaOH (1N), 10 times with 1 mL of 50 mM sodiumbicarbonate, and then 5 times each with water, THF, and dichloromethane.

5. Cleavage from Resin

1% trifluoroacetic acid (TFA) in 1 mL DCM is added to the resin twicefor 1 minute each. The supernatant is collected and DCM evaporated. Theresulting oily film is rehydrated in H₂O and lyophilized, resulting in alight yellow powder. An NMR is taken to confirm the presence of the PEGpolymer.

6. Coupling to Polymer

Folic acid-linker is reacted with 6 equivalents of a cyclodextrincopolymer (oxidized as in Example 10) by mixing in 50 mmol borate (pH8.5). The reaction mixture is analyzed and conjugation polymer confinedby a GPC system with a UV detection at 285 nm.

Example 18

Folate Ligand Attachment to Cyclodextrin Polymer

1. Coupling

36 mg of t-butyl carbazate dissolved in 240 μL of DCM/ethyl acetate(1:1) was added to 260 mg of FMOC-PEG₃₄₀₀-NHS (Shearwater Polymers) andmixed at room temperature for 2 hours. The product was precipitated twotimes from ethyl acetate/ether (1:1).

2. Removal of Protecting Group

FMOC protecting group was removed with 20% piperidine in DMF. Thesolvent was removed in vacuo and product redissolved in 1.3 mL of DMSO.

3. Folic Acid Coupling

1.2 equivalents of folic acid and DCC and one drop of pyridine was thenadded and the resulting solution stirred in the dark at room temperaturefor 6 hours. DMSO was removed in vacuo and conjugation of folic acid wasconfirmed by GPC with UV monitoring at 285 nm.

4. Removal of Hydrazide Protecting Group

Finally, the hydrazide was deprotected by stirring in 4M HCl in dioxanefor 1 hour before removing the solvent in vacuo. The final product waspurified by Toyopearl HW-40F column chromatography.

5. Coupling to Polymer

Folic acid-linker is reacted with 6 equivalents of a cyclodextrincopolymer (oxidized as in Example 10) by mixing in 50 mmol borate (pH8.5). The reaction mixture is analyzed and conjugation polymer confirmedby a GPC system with a UV detection at 285 nm.

Example 19

Transferrin Ligand Attachment to Cyclodextrin Polymer

1. Transferrin Oxidation

500 mg of iron-free human transferrin (Sigma of St. Louis, Mo.) isdissolved in 30 mM sodium acetate buffer and cooled to 0° C. To thissolution is added 20 mg of sodium periodate dissolved in 4 μL of 30 mMsodium acetate. The mixture is stirred at 0° C. overnight. Next 1 g ofAG501-X8 resin (Biorad) is added to remove salts before the solution islyophilized.

2. Resin Coupling

20 mg of FMOC-PEG₃₄₀₀-NHS (Shearwater Polymers, Inc. of Huntsville,Ala.) was dissolved in 0.5 mL of anhydrous N,N-dimethylformamide (DMF)and added to 10 equivalents of hydrazide 2-chlorotrityl resin(Novabiochem USA of La Jolla, Calif.) swelled in DMF. The mixture wasstirred at 60° C. until all the polymer was coupled to the resin, asdetermined by a GPC system equipped with an ultraviolet (UV) detector.The resin-polymer was then transferred to a sintered glass column forall further reactions.

3. Resin Capping

The unreacted hydrazide groups on the resins were capped with aceticanhydride and the acetic acid products were neutralized bydiisopropylethylamine.

4. Removal of Protecting Group

The FMOC protecting group was removed by two washes with 20% piperidinein DMF (1 mL total volume). The resin was then washed 10 times with 1 mLDMF and 5 times with 1 mL H₂O.

5. Transferrin Coupling

To the resin is added 1.2 equivalents of transferrin dissolved in 0.05 Msodium carbonate and 0.1 M sodium citrate buffer, pH 9.5. 5 Mcyanoborahydride in 1N NaOH is then added to the solution. The glasscolumn is placed on a rotator and mixed for 2 hours. The resin is thenwashed 15 times with water and 5 times each with tetrahydrofuran (THF)and DCM.

6. Cleavage from Resin

1% trifluoroacetic acid (TFA) in 1 mL DCM is added to the resin twicefor 1 minute each. The supernatant is then collected and DCM evaporated.The resulting oily film is rehydrated in H₂O and lyophilized.

7. Coupling to Polymer

Transferrin linker is reacted with 6 equivalents of a cyclodextrincopolymer by reductive animation with sodium cyanoborohydride: first,the copolymer is added to transferrin linker dissolved in 0.05 M sodiumcarbonate and 0.1 M sodium citrate buffer. 5 M cyanoborohydride in 1NNaOH is added and the reaction is stirred for 2 hours at roomtemperature. Unreacted aldehyde sites are blocked by adding ethanolamineand reacting for 15 minuted at room temperature. The resulting conjugateis purified by dialysis.

Example 20

General Procedure for Cyclodextrin Copolymer Complexation with SmallMolecules

Cyclodextrin-based copolymer (CD-polymer) is dissolved in water, buffer,or organic solvent at the appropriate concentration. The small moleculeis dissolved in a solvent miscible with the solvent of the CD-polymersolution and is added to the CD-polymer solution. The mixture is thenstirred for ½ hour and then allowed to come to equilibrium overnight.

Example 21

Cyclodextrin Copolymer Complexation with Doxorubicin

Doxorubicin and CD-polymer were dissolved at various concentrations inPBS (phosphate buffered saline, pH 7.2). The association constantbetween the CD and doxorubicin was determined by measuring the extent ofdoxorubicin's fluorescence increase upon complexation with the CD. (Thehydrophobic interaction between the CD and doxorubicin enhances thefluorescence intensity). Association constant was approximately 200 M⁻¹at pH 7.1. Addition of β-CD consistently enhanced doxorubicinfluorescence, indicating complexation between the CD-polymer anddoxorubicin. Husain et al., Applied Spectroscopy Vol. 46, No. 4, 652-658(1992) found the association constant between β-CD and doxorubicin to be210 M⁻¹ at pH 7.1.

Example 22

Small Molecule Delivery to Cultured Cells

Media containing doxorubicin and doxorubicin/CD-polymer complexes atvarious concentrations were applied to cultured cell lines. After 5hours, the media was removed and replaced with fresh media. Doxorubicineffect on cell survival was determined by the MTT([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium) toxicityassay. (R. Ian Feshney, “Culture of Animal Cells”, 3rd ed.,Wiley-Liss:New York (1994)). The results are illustrated in the tablebelow. Copolymer 15 or 16 (138 μM equivalent of CD monomer) was nottoxic to KB or KB-VI (a multidrug resistant derivative of KB) cell linesin the absence of doxorubicin. For receptor-mediated delivery, a ligandsuch a folate is covalently attached to the CD-polymer used fordoxorubicin complexation.

IC₅₀ (μM of Cell Line CD-polymer doxorubicin) KB none  ˜0.1 KB-VI(multidrug resistant) none ˜10 KB-VI copolymer 15 or 16 (138 μM  ˜2-3equivalent of CD monomer)

Example 23

Fixed Permanent Charged Copolymer Complexation with Plasmid

In general, equal volumes of fixed charged CD-polymer and DNA plasmidsolutions in water are mixed at appropriate polymer/plasmid chargeratios. The mixture is then allowed to equilibrate and self-assemble atroom temperature overnight. Complexation success is monitored bytransferring a small aliquot of the mixture to 0.6% agarose gel andchecking for DNA mobility. Free DNA travels under an applied voltage,whereas complexed DNA is retarded at the well.

1 μg of DNA at a concentration of 0.2 μg/μL in distilled water was mixedwith 10 μL of copolymer 15 at polymer amine: DNA phosphate charge ratiosof 2, 4, 6, 12, 24, 36, 60, and 120. The solution was mixed manually bya micropipette and then gently mixed overnight on a lab rotator. 1 μg/μLof loading buffer (40% sucrose, 0.25% bromophenol blue, and 200 mMTris-Acetate buffer containing 5 mM EDTA (Gao et al., Biochemistry35:1027-1036 (1996)) was added to each solution the following morning.Each DNA/polymer sample was loaded on a 0.6% agarose electrophoresis gelcontaining 6 μg of EtBr/100 mL in 1×TAE buffer (40 mM Tris-acetate/1 mMEDTA) and 40V was applied to the gel for 1 hour. The extent ofDNA/polymer complexation was indicated by DNA retardation in the gelmigration pattern. The polymer (15) retarded DNA at charge ratios of 6and above, indicating complexation under these conditions.

Example 24

Crosslinking Copolymer Complexation with Plasmid

Copolymer 15 or copolymer 16 is oxidized as in Example 10. Oxidizedcopolymer 15 or 16 is then complexed with a DNA plasmid as in Examples23 and 26. A crosslinking agent (for example, PEG₆₀₀-Dihydrazide) isthen added to encapsulate the DNA. Encapsulation success is determinedby light scattering and visualized by electron microscopy.

Example 25

Variably Charged (pH-sensitive) Copolymer Complexation with Plasmid

Equal volumes of a CD-polymer and DNA plasmid solutions in water aremixed in appropriate polymer/plasmid charge ratios. The pH of themixture is adjusted to form a charged CD-polymer. The mixture is thenallowed to equilibrate and self-assemble at room temperature for 30minutes. A crosslinking agent (for example, PEG₆₀₀-Dihydrazide) is thenadded to encapsulate the DNA. A concentrated buffer solution is thenadded to render the pH and thus the CD-polymer neutral. Encapsulationsuccess is determined by light scattering and visualized by electronmicroscopy.

Example 26

Transfection Studies with Plasmids Encoding Luciferase Reporter Gene

BHK-21 cells were plated in 24 well plates at a cell density of 60,000cells/well 24 hours before transfection. Plasmids encoding theluciferase gene were encapsulated by the CD-polymer as in Examples 23 or25 such that the DNA/polymer complexes were assembled at polymer amine:DNA phosphate charge ratios of 6, 12, 24, 36, and 60 as described in DNAbinding studies of Example 23. Media solution containing the DNA/polymercomplexes was added to cultured cells and replaced with fresh mediaafter 5 hours of incubation at 37° C. The cells were lysed 48 hoursafter transfection. Appropriate substrates for the luciferase lightassay were added to the cell lysate. Luciferase activity, measured interms of light units produced, was quantified by a luminometer. Theresults are shown in FIG. 1A. DNA/polymer complexes successfullytransfected BHK-21 cells at a charge ratios of 6, 12, and 24. Celllysate was also used to determine cell viability by the Lowry proteinassay. (Lowry et al., Journal of Biological Chemistry, Vol. 193, 265-275(1951)). The results are shown in FIG. 1B. Maximum toxicity was seen ata polymer amine: DNA phosphate charge ratios of 36 and 60 with 91% cellsurvival.

Example 27

Transfection Studies with Plasmids Encoding Luciferase Reporter Gene

BHK-21 cells were plated in 24 well plates at a cell density of 60,000cells/well 24 hours before transfection. Plasmids encoding theluciferase gene were encapsulated by the CD-polymer as in Example 23except copolymer 15 was replaced with copolymer 16 and that theDNA/polymer complexes successfully transfected BHK-21 cells at chargeratios of 10, 20, 30, and 40 with maximum transfection at polymeramine:DNA phosphate charge ratio of 20. Media solution containing theDNA/polymer complexes was added to cultured cells and replaced withfresh media after 24 hours of incubation at 37° C. The cells were lysed48 hours after transfection. Appropriate substrates for the luciferaselight assay were added to the cell lysate. Luciferase activity, measuredin terms of light units produced, was quantified by a luminometer. Theresults are shown in FIG. 1A. DNA/polymer complexes successfullytransfected BHK-21 cells at a charge ratios of 6, 12, and 24. Celllysate was also used to determine cell viability by the Lowry proteinassay. (Lowry et al., Journal of Biological Chemistry, Vol. 193, 265-275(1951)). The results are shown in FIG. 1B. Maximum toxicity was seen ata polymer amine: DNA phosphate charge ratios of 40 and 50 with 33% cellsurvival.

Example 28

Transfection Studies with Plasmids Encoding GFP Reporter Gene

Plasmids encoding the green fluorescent protein are encapsulated by theCD-polymer as in Examples 23 or 25. Media solution containing theDNA/polymer complexes is added to cultured cells and replaced with freshmedia after 5 hours of incubation at 37° C. The cells are detached fromthe surface with trypsin, washed, and resuspended in Hanks Balanced SaltSolution with propidium iodide. The cells are then analyzed byfluorescence activated cell sorting (FACS). Cell viability is determinedby cell size and propidium iodide exclusion, and transfection success byGFP protein fluorescence.

Example 29

Polymer Complexation with Oligos

Complexation with antisense oligos is accomplished following theprocedures for plasmid complexation of Examples 23 or 25.

Example 30

Transfection Studies with Oligos

Antisense oligos directed against the luciferase gene are encapsulatedby the CD-polymer as described in Example 29. Media solution containingthe oligo/polymer complexes is added to HeLa X1/5 cells (HeLa cells thatconstitutively express the luciferase gene, donated by CLONTECH) andreplaced with fresh media after 5 hours of incubation at 37° C. Cellsare lysed 48 hours after transfection and appropriate substrates for theluciferase assay are added to the lysates. Luciferase activity, measuredin terms of light units produced, is quantified by a luminometer.Transfection success is determined by knockout of luciferase activity.

Example 31

Toxicity of β-cyclodextrin(cystamine)-DTBP copolymer, 15

The acute toxicity of copolymer 15 was investigated using Swiss-Webster“white mice.” A total of 48 mice were used as described in the tablebelow. Single intravenous (i.v.) or intraperitoneal (i.p.) injections ofsterile saline solutions or of copolymer 15 were given to the mice. Theanimals were followed for five days after which they sacrificed andgross necropsy performed. No mortality and no toxicity was observed.

Concen- Dose Group #/Sex tration Volume Dose Treatment No. (M/F)Copolymer (mg/mL) (mL) (mg) Regimen 1 3/3 CoPolymer 15 0.5275 0.1 0.05i.v., once 2 3/3 CoPolymer 15 5.275 0.1 0.53 i.v., once 3 3/3 CoPolymer15 52.75 0.1 5.28 i.v., once 4 3/3 CoPolymer 15 0.5275 0.1 0.05 i.p.,once 5 3/3 CoPolymer 15 5.275 0.1 0.53 i.p., once 6 3/3 CoPolymer 1552.75 0.1 5.28 i.p., once 7 3/3 0.9% saline 0.000 0.1 0.00 i.v., once 83/3 0.9% saline 0.000 0.1 0.00 i.p., once

Example 32

Transfection Studies with Plasmids Encoding Luciferase Reporter Gene

Plasmids encoding the luciferase gene were encapsulated by theCD-polymer as in Example 23 except copolymer 15 was replaced withcopolymer 16. The DNA/polymer complexes were use to successfullytransfect BHK-21 or CHO-K1 cells, each plated in 24 well plates at acell density of 60,000 cells/well 24 hours before transfection, atvarious charge ratios in 10% serum and serum-free conditions followingthe procedure outlined in Example 27. The cells were lysed 48 hoursafter transfection. Appropriate substrates for the luciferase lightassay were added to the cell lysate. Luciferase activity, measured interms of light units produced (i.e., relative light units (RLU)), wasquantified by a luminometer. Cell lysate was also used to determine cellviability by the Lowry protein assay. (Lowry et al., Journal ofBiological Chemistry, Vol. 193, 265-275 (1951)). Toxicity was measuredby determining total cellular protein in the wells 48 hours aftertransfection. The transfection and cell survival results in 10% serumand serum free media are shown in FIGS. 2A and 2B.

Luciferase protein activity in BHK-21 cells transfected in serum-freeconditions reached a stable maximum at 30+/− with ˜5×10⁷ RLUs. Thepresence of 10% serum in the transfection media decreased luciferaseactivity at all charge ratios except 70+/−. With CHO-K1 cells,increasing charge ratio also enhanced the transfection for allconditions tested. Additionally, transfection in serum decreased lightunits by an order of magnitude.

Copolymer 16 showed toxicity only to BHK-21 cells for transfections inthe absence of serum. Toxicity was minimized with the presence of 10%serum during transfection. No noticeable toxicity was observed fromtransfections to CHO-K1 cells.

Following the procedure of Example 32, transfection efficiency andtoxicity of various non-viral vectors with BHK-21 and CHO-K1 cells werestudied and compared against those achieved with DNA/copolymer 16complexes. The BHK-21 and CHO-K1 cells were transfected at a range ofcharge ratios and starting cells densities for all vectors in serum-freemedia. The results are shown in FIGS. 3A and 3B and illustrate theoptimum transfection conditions found for each vector.

It should be understood that the foregoing discussion and examplesmerely present a detailed description of certain preferred embodiments.It will be apparent to those of ordinary skill in the art that variousmodifications and equivalents can be made without departing from thespirit and scope of the invention. All the patents, journal articles andother documents discussed or cited above are herein incorporated byreference.

1. A water-soluble, linear cyclodextrin copolymer comprising repeatingunits of formula VIa, VIb, or both:

wherein C is a substituted or unsubstituted oxidized cyclodextrinmonomer and A is a comonomer bound to cyclodextrin C.
 2. A cyclodextrincopolymer of claim 1, wherein said cyclodextrin monomer is an α-, β-,γ-cyclodextrin, or combination thereof.
 3. The copolymer of claim 1,wherein A is selected from: —HNC(O)(CH₂)_(x)C(O)NH—,—HNC(O)(CH₂)_(x)SS(CH₂)_(x)C(O)NH—, —⁺H₂N(CH₂)_(x)SS(CH₂)_(x)NH₂ ⁺—,—HNC(O)(CH₂CH₂O)_(x)CH₂CH₂C(O)NH—,—HNNHC(O)(CH₂CH₂O)_(x)CH₂CH₂C(O)NHNH—, —⁺H₂NCH₂(CH₂CH₂O)_(x)CH₂CH₂CH₂NH₂⁺—, —HNC(O)(CH₂CH₂O)_(x)CH₂CH₂SS(CH₂CH₂O)_(x)CH₂CH₂C(O)NH—, —HNC(NH₂⁺)(CH₂CH₂O)_(x)CH₂CH₂C(NH₂ ⁺)NH—, —SCH₂CH₂NHC(NH₂ ⁺)(CH₂)_(x)C(NH₂⁺)NHCH₂CH₂S—, —SCH₂CH₂NHC(NH₂ ⁺)(CH₂)_(x)SS(CH₂)_(x)C(NH₂ ⁺)NHCH₂CH₂S—,—SCH₂CH₂NHC(NH₂ ⁺)CH₂CH₂(OCH₂CH₂)_(x)C(NH₂ ⁺)NHCH₂CH₂S—,

where x=1-50 and y+z=x.
 4. The copolymer of claim 1, wherein A isbiodegradable or acid-labile.
 5. The copolymer of claim 1, wherein thecyclodextrin copolymer is crosslinked to a polymer.
 6. The copolymer ofclaim 5, further comprising wherein at least one ligand is bound to thelinear cyclodextrin copolymer; wherein said ligand allows the copolymerto target and bind to a cell.
 7. The copolymer of claim 1, furthercomprising wherein at least one ligand is bound to the linearcyclodextrin copolymer; wherein said ligand allows the copolymer totarget and bind to a cell.
 8. The copolymer of claim 1, whereinsubstantially all of the repeating units are of formula VIa, VIb, orboth.
 9. The copolymer of claim 1, wherein all of the repeating unitsare of formula VIa, VIb, or both.
 10. A therapeutic compositioncomprising the copolymer of claim 1, 5, 6, or 7 and a therapeutic agent.11. A method of preparing a water-soluble linear oxidized cyclodextrincopolymer, comprising: oxidizing a water-soluble, linear cyclodextrincopolymer having repeating units of formula Ia, Ib, or a combinationthereof:

wherein C′ is a substituted or unsubstituted cyclodextrin monomer and Ais a comonomer bound to cyclodextrin monomer C′.
 12. The method of claim11, further comprising reacting said water-soluble, linear oxidizedcyclodextrin copolymer with a ligand to form a water-soluble, linearoxidized cyclodextrin copolymer having at least one ligand bound to thecopolymer, wherein said ligand allows the copolymer to target and bindto a cell.
 13. A method of preparing a water-soluble, linear oxidizedcyclodextrin copolymer, comprising: (a) iodinating an oxidizedcyclodextrin monomer precursor to form an oxidized diiodinatedcyclodextrin monomer precursor formula VIIa, VIIb, VIIc or a mixturethereof:

(b) copolymerizing said oxidized diiodinated cyclodextrin monomerprecursor with a comonomer A precursor to form a linear oxidizedcyclodextrin copolymer having a repeating units of formula VIa, VIb, ora combination thereof:

wherein C is a substituted or unsubstituted oxidized cyclodextrinmonomer and A is a comonomer bound to the cyclodextrin comonomer C. 14.The method of claim 13, further comprising reacting said water-soluble,linear oxidized cyclodextrin copolymer with a ligand to form awater-soluble, linear oxidized cyclodextrin copolymer having at leastone ligand bound to the copolymer, wherein said ligand allows thecopolymer to target and bind to a cell.
 15. The method of claim 13,further comprising aminating said oxidized diiodinated cyclodextrinmonomer precursor to form an oxidized diaminated cyclodextrin monomerprecursor of formula VIIIa, VIIIb, VIIIc or a mixture thereof:

copolymerizing said oxidized diaminated cyclodextrin monomer precursorwith a comonomer A precursor to form said water-soluble linear oxidizedcyclodextrin copolymer having repeating units of formula VIa, VIb, or acombination thereof.
 16. The method of claim 13, further comprisingaminating said oxidized diiodinated cyclodextrin monomer precursor toform an oxidized diaminated cyclodextrin monomer precursor of formulaIXa, IXb, IXc, or a mixture thereof:


17. A method of producing a crosslinked cyclodextrin polymer comprising:reacting at least one water-soluble, linear cyclodextrin copolymerhaving repeating units of formula VIa, VIb or a combination thereof:

where C is a substituted or unsubstituted oxidized cyclodextrin monomerand A is a comonomer bound to cyclodextrin monomer C, with a polymer inthe presence of a crosslinking agent.
 18. The method of claim 17,wherein said polymer is a water-soluble, linear oxidized cyclodextrincopolymer.
 19. A method of delivering a therapeutic agent comprisingadministering a therapeutically effective amount of the therapeuticcomposition of claim 10.